We are continuing to develop procedures for unambiguously identifying those serine and threonine residues of glycopeptides that have glycosyl chains attached. In this approach, the glycopeptide is digested with pronase, which cleaves the peptide backbone in a random fashion. The digestion mixtures are analyzed by MALDI-MS after digestion for 5, 10, and 15 min. These spectra contain a series of peptide and glycopeptide peaks that identify the glycosylation site. The primary advantages of our pronase digestion/MALDI-TOF procedure for identifying glycosylation sites are its speed (less than 30 min) and sensitivity (requiring only 1-10 picomoles of glycopeptide). Furthermore, this approach is suitable for determining the sites of other post-translational modifications, including phosphorylation, sulfation, disulfide bonds, and acetylation. This project is aimed at filling the current void of analytical procedures capable of determining O-linked glycosylation sites fro m sub-nanomole quantities of a glycopeptide.